Research in Dr. C.D. Atreya's laboratory primarily focuses towards conducting CBER mission-related research pertaining to the pediatric RNA viral vaccine safety. The emphasis is on molecular host-virus interaction(s) pertinent to the vaccine-associated adverse reactions with reference to rotavirus and rubella virus as well as certain aspects of influenza virus-host interactions relevant to the virus growth (in collaboration with Dr. Levandowski) and vaccinia-based smallpox vaccine-host pertinent to neurotoxicity (in collaboration with Dr. Carbone). Ongoing research projects include identification and characterization of interaction of non-immune cellular proteins with specific proteins from each virus (rotavirus, rubella virus, influenza virus and vaccinia-based smallpox vaccines) using a number of molecular techniques including yeast two-hybrid system based screening of human, simian and mouse (adult and neonatal as appropriate) cDNA libraries, cellular overexpression of interactive proteins and utilization of such cell lines in evaluation of viral replication and pathogenesis and vaccine associated adverse reactions, as well as confocal microscopy following immunostaining. Vaccine safety: Molecular mechanisms of live, attenuated rubella virus vaccine adverse reactions. Most common complications associated with rubella virus (RV) vaccine occur in adult women and are manifested in polyarthraliga and arthritis, possibly due to persistence and localized viral replication. In addition, in utero fetal infection by RV in the first trimester of pregnancy can cause fetal teratogenesis. Based on the guidelines developed after 1996 IOM meeting, it has been recognized that there is a need for elucidating the mechanisms of adverse reactions associated with the RV vaccine as well as basic understanding of how the virus induces teratogenesis. In the past, we have shown: a) cause of adverse reactions with live virus vaccine could probably be due to interaction of host proteins with RV. b) The host proteins, calreticulin and La (known autoantigens) interact with RV RNA and patients with natural RV infection develop significant anti-La response following prolonged infection. c) Cellular calreticulin chaperones rubella virus E1 glycoprotein and mutations in the E1 glycosylation sites affect viral assembly and release due to perturbations in E1 chaperoning to proper sites of viral assembly. d) A host protein, p32, which is the receptor of complement 1 protein's (C1q) "globular head" as rubella virus/vaccine capsid protein binder. Perturbations in p32 functions have been implicated in SLE-associated arthritis. By anology, RV capsid-p32 interaction could play a major role in inducing transient/chronic arthritis associated with rubella virus vaccine as well as with natural infections. e) Rubella virus non-structural proteins have domains which interact with cell cycle regulatory protein i.e. retinoblastoma protein (RB) both in vivo and in vitro. This interaction could result in the alteration in cell growth upon RV infection and may lead to the RV induced teratogenesis. PROGRESS. We identified a host protein, Citron-Kinase (CK), a cellular protein that regulates cytokinesis (cell division) as rubella virus replicase protein ligand and demonstrated that rubella virus and the replicase protein alone induce tetraploid status in cell culture via CK interaction that have implications in understanding the virus induced teratogenesis. Vaccine Efficacy : Studies on rotavirus replication, pathogenicity, potency and host range. Rotavirus is a 70 nm non-enveloped virus containing 11 segments of double stranded RNA genome. Severe gastroenteritis in both developed and less developed countries is caused by rotaviruses and currently there is no licensed vaccine available, after the first licensed rhesus-human reassortant tetravalent live attenuated rotavirus vaccine has been withdrawn. Disease aspect of rotavirus results from rapid replication of the virus in intestinal cells, outstripping the ability of the intestine to replace damaged cells. Thus virus-host molecular interactions play a crucial role in disease outcome. The research focus of this project is therefore to identify and study rotavirus protein-host protein interactions relevant to viral replication and pathogenesis. Past activities 1. Lab was set-up to perform rotavirus potency testing for the first licensed vaccine by plaque-assay method. Several vaccine lots have been released based on in-house potency testing and by review of submitted lot release protocols, until the product was withdrawn. 2.We have produced novel mutant forms of human rotavirus NSP4 that have potential in using as subunit toxoid vaccines. Studies are underway to fully characterize the mutants. In addition, we have cloned and sequenced several important rotavirus strain-specific genes (VP4, VP5, VP8, NSP4, NSP5 and NSP6 to identify their role in viral replication and pathogenesis. 3. Recently we identified a peroxisomal targeting signal tri-peptide in rotaviral VP4 protein. This is a seminal discovery to the entire virology field, since functional peroxisomal targeting tri-peptide sequences were not reported before in any viral proteins. Since peroxisomal biogenesis associated disorders were known, we are currently pursuing the idea that the rotaviral protein targeting to the organelle may have serious implications in viral pathogenesis and possibly associated with the vaccine induced adverse reactions in infants. PROGRESS 1. Discovered peroxisomal targeting signals in the proteins of several medically important viruses. Given that peroxisomes are the cellular sites of cholesterol metabolism, toxin degradation and gluconeogenesis and several enveloped viruses need lipid rafts for assembly and budding, our finding of viral protein targeting to the organelle have an impact on understanding viral pathogenesis and evading mechanisms and assembly. 2. Viroplasms/viroplasm-like structures (VLS) are the sites of rotaviral replication and viral protein assembly. VLS is believed to be formed due to NP5/NSP2 synergistic effect. We have recently identified and demonstrated the critical determinants of the rotavirus NSP5 alone that are responsible for VLS, independent of NSP2. This has changed the way we previously understood the NSP5 functions and is a significant contribution to the field of rotavirus replication and intracellular viral protein assembly. 3. Characterized the enterotoxin gene from a human rotavirus vaccine strain (Chinese), the only live rotavirus vaccine available to prevent rotavirus disease that has been licensed in China. Smallpox Vaccine Safety: Development and Validation of Pre-Clinical Toxoclogy Tests for Vaccinia-based Smallpox Vaccine through Molecular Mechanism of Vaccine Neurotoxology. Smallpox virus is a Category A bioterrorism/biowarfare agent. The licensed vaccinia virus-based vaccine (Dryvax) is effective, but produces significant and serious adverse events (AE) (Lane, 1969; Vega, 1969; Adams, 1973; Terzin, 1974; Edis, 1975; Gurvich, 1975; Kurata, 1977); one of the most deadly AEs is central nervous system (CNS) disease, with children being at greater risk than adults. Fortunately, new, and, it is hoped, safer smallpox vaccines are in development. As a standard regulatory consideration, pre-clinical neurotoxicity assays are used as in an attempt to predict the risk of damage to the human CNS (neurotoxicity) from live virus vaccines; no such validated neurotoxicity assay is available for vaccinia-based smallpox vaccines. To avoid the expense and validation problems inherent in primate testing, we have developed a prototype smallpox vaccine neurotoxicity assay using rodents. Preliminary data indicate this assay can discriminate differences in vaccinia virus strain-specific neurotoxicity among smallpox vaccines (Dryvax, Lister), and laboratory strains (WR, MVA), with WR>Lister>Dryvax>MVA in order of decreasing neuro-toxicity. Here we propose to 1) complete development and validation of the in vivo mouse neurotoxicity assay as a standardized regulatory safety test to expedite the licensing of safer smallpox vaccines, and 2) use this assay as a disease model to study the molecular pathogenesis of vaccinia-based smallpox vaccine neurotoxicity by identifying critical virus-neural cell gene/gene-product interactions. These studies will improve smallpox vaccine safety tests, e.g., small animal, in vitro and molecular biological-based neurotoxicity assays, and can promote new vaccine development, and e.g., rational attenuation of smallpox vaccines via targeted mutations. PROGRESS. We have developed and tested a neonatal mouse model for assessing the relative toxicity of vaccinia virus strains. We have also made progress towards achieving the second objective of the project by identifying the viral and host gene interactions in adult and infant using human brain tissue-derived cDNA libraries. We are currently assessing the functional role of these interactions. This project incorporates FY2002 projects 1Z01BK002012-06, 1Z01BK002013-06, and 1Z01BK002021-01.